The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
Solid-phase synthesis is a well-known and widely spread technology in modern organic chemistry for preparation of various biologically important compounds. It has found many new applications such as combinatorial libraries and the peptide-based DNA analogue (PNA, peptide nucleic acids). Along with the technology development, the need for labeling those biologically interesting compounds during solid-phase synthesis has appeared. Several non-fluorescent labeling candidates have been published (Arya, R. and Gariepy, J., 1991, Bioconjugate Chem., 2, 323, Cuenoud, B. and Schepartz, A., 1991, Tetrahedron, 47, 2535, Rana, T. M., Ban, M. and Hearst, J. E., 1992, Tetrahedron Lett., 33, 4521, Song, A. I. and Rana, T. A., 1997, Bioconjugate Chem., 8, 249). These labels have been developed mainly to incorporate metal ions such as Fe, Cu in a peptide to act as sequence-specific cleaving agents for proteins. Radioactive .sup.153 Gd, .sup.111 In etc. can be used for in vivo tumor localization and radioimmunotherapy. Using lanthanide ions and a separate luminescence enhancement step these published monomers can be used as luminescent labels but a separate enhancement step is needed to produce the luminescence to be detected. During the luminescence enhancement the lanthanide has to be dissociated from the label and then spatial information will be lost.
Fluorescent label monomers for solid-phase synthesis have been published (Lohse, J., Nielsen, P. E., Harrit, N. and Dahl, O., 1997, Bioconjugate Chem., 8, 503, McCafferty, D. G., Bishop, B. M., Wall, C. G., Hughes, S. G., Mecklenberg, S. L., Meyer, T. J. and Erickson, B. W., 1995, Tetrahedron, 51, 1093, WO 96/03409). Fluorescein and other organic chromophores were used in those studies. However, such labels and labeled biomolecules suffer from many commonly known drawbacks such as Raman scattering, other fluorescent impurities, low water solubility, concentration quenching etc.
In the specific binding assays, such as e.g. immunoassays, DNA hybridization assays, receptor-binding assays, and cellular binding assays, generally very low concentrations of the analytes to be measured are present. Therefore, various labeling compounds have been developed that allow the labeled reactant to be detected and quantitated with high sensitivity. In immunoassays and DNA hybridization assays, time-resolved luminescence spectroscopy using lanthanide chelates is well known. (e.g. I. Hemmila, T. Stahlberg, and P. Mottram (eds.), "Bioanalytical Applications of Labeling Technologies", Wallac, Turku, 1994). Stable photoluminescent (in this context simply referred to as luminescent) lanthanide chelates also have other applications, e.g. fluorescence microscopy and cytometry. Therefore, a number of attempts have been made to develop new highly luminescent chelate labels suitable for those types of time-resolved fluorometric applications. These include e.g. stable chelates composed of derivatives of pyridines (U.S. Pat. No. 4,920,195, U.S. Pat. No. 4,801,722, U.S. Pat. No. 4,761,481, PCT WO FI-91/00373, U.S. pat. appl. Ser. No. 08/135,525, EP Appl. 0770610, Remuinan, M. J., Roman, H., Alonso, M. T. and Rodriguez-Ubis, J. C., 1993, J. Chem. Soc., Perkin Trans.2, 1099), bipyridines (U.S. Pat. No. 5,216,134), terpyridines (U.S. Pat. No. 4,859,777, U.S. Pat. No. 5,202,423, U.S. Pat. No. 5,324,825) or various phenolic compounds (U.S. Pat. No. 4,670,572, U.S. Pat. No. 4,794,191, Ital. Pat. 42508 A/89) as the energy mediating groups and polycarboxylic acids as chelating parts. In addition, various dicarboxylate derivatives (U.S. Pat. No. 5,032,677, U.S. Pat. No. 5,055,578, U.S. Pat. No. 4,772,563), macrocyclic cryptates (U.S. Pat. No. 4,927,923, PCT WO 93/5049, EP-A493,745) and macrocyclic Schiff bases (EP-A369,000) have been patented. None of these patents and articles present any methods for labeling of a biospecific binding reactant such as a hapten, a peptide, a receptor ligand, a drug or PNA oligomer, with luminescent labels by using solid-phase synthesis.